Integral membrane proteins constitute approximately 30% of all genes, making them a very important class of proteins. In addition, more than half of all current drug targets are membrane proteins. While the rate of structure determinations of soluble proteins has dramatically accelerated over the last decade, that of membrane proteins has lagged far behind, reflecting the technical challenges associated with working with this class of proteins. The enzymes DsbA and DsbB comprise the functional machinery in E. coli and many other bacteria to catalyze the formation of disulfide bonds in proteins in the periplasmic space. DsbA is the direct oxidant of protein substrates and resides in the periplasm. DsbB is an integral membrane enzyme which reoxidizes DsbA for further catalysis and transfers reducing equivalents to a bound quinone. Homologs of DsbB have been identified in a large number of different bacterial genomes, with a topological and functional characterization into 3 different groups. Current classes of antibiotics, as a result of actual killing of the bacteria, create an evolutionary pressure for bacteria to evolve to escape their effects. An alternative approach has been proposed which targets bacterial virulence. In this approach, the goal is to inhibit specific bacterial functions that promote infection and are essential to persistence such as binding, invasion, subversion of host defenses, and chemical signaling. Strikingly, disulfide bond formation is critical for the functioning of many proteins that mediate virulence functions, therefore the Dsb proteins have been suggested to be appropriate targets for the development of anti-virulence agents. These results clearly support efforts to develop small molecule inhibitors of DsbB, as well as its functional homologs, as a novel approach to inhibit bacterial virulence. We are proposing 3 Aims to provide additional structural insights into the DsbB family of proteins and develop novel small molecule inhibitors of DsbB as a novel approach to inhibiting bacterial virulence: Aim 1: Refined structure of DsbB with definition of sidechains. In this Aim, we will employ novel NMR approaches to determine the conformations of the sidechains in E. coli DsbB. This information is critical to a more detailed understanding of the mechanism of the enzyme. Aim 2: Development of small molecule inhibitors of DsbB. In this Aim, we will follow up on several hits identified in an NMR based screen to develop potent small molecule inhibitors of DsbB function. The effect of these on DsbB activity and E. coli virulence functions will be assessed. Aim 3: Structure determination of Francisella tularensis Group II DsbB. As there is currently no structural information for the Group II class of DsbB enzymes, we are proposing to solve the structure of the Group II DsbB from Francisella tularensis, a potent human pathogen that is the causative agent for tularemia. PUBLIC HEALTH RELEVANCE: DsbB is an important protein for the functioning of many proteins that mediate the ability of bacteria to enter human cells, inject material into those cells, and to release toxins. All these functions contribute to the effects of bacterial infections on humans. To better understand the protein, we are working to determine the structure of two types of DsbB proteins from human pathogens. If we can inhibit the function of DsbB, we will have a new approach to treat bacterial infections. To that end, we are proposing to develop inhibitors of DsbB, a new potential approach to treating bacterial infections.